Insight into IGF-1R binding and activation using novel recombinant and synthetic IGF-II FRET probes

J Cottam Jones1, P Harris2, D Scanlon1, M Brimble2, A Abell1 and B Forbes3

  1. The University of Adelaide, Adelaide 5001, SA, Australia
  2. The University of Auckland, Auckland 1010, New Zealand
  3. Flinders University of South Australia, Bedford Park, 5042, SA, Australia

While invaluable understanding has been gained from structures of insulin (and more recently IGF-I) in complex with fragments of the insulin receptor (IR)(1), the mechanism of ligand engagement with the receptor (or with the highly similar IGF-1R) still is not entirely understood. We took a fluorescence resonance energy transfer (FRET) approach to probe for sites of interaction between IGF-II and the IGF-1R using coumarin (Cou) fluorescent probes within the classical binding site 1 and in site 2 of IGF-II, and endogenous Trp residues of IGF-1R as the FRET partner. Insulin receptor structural change has previously been monitored by a change in Trp fluorescence, suggesting that Trp may lie in close proximity to the ligand binding pockets(2). Analysis of available insulin:IR structures (and more recently IGF-I:IR/IGF-1R structures) revealed that several Trp residues could act as potential FRET partners with IGF-II site 1 and site 2 residues. Thus Phe19Cou IGF-II and Phe28Cou IGF-II proteins, with substitutions in site 1 and site 2 respectively, were produced using convergent synthesis or recombinant methods to incorporate the non-canonical Cou amino acid. These fluorescent analogues bind with nanomolar affinities to the IGF-1R. We demonstrated both are good probes for investigating the binding interactions of IGF-II with the IGF-1R and the outcomes of the FRET analyses will be discussed.
References: 1. Ward et al. (2013) BioEssays 35(11):945-954. 2. Lee J, Pilch PF, Shoelson SE, & Scarlata SF (1997) Biochemistry 36(9):2701-2708.